Laboratory Methods in Enzymology: Protein Part B
- 1st Edition, Volume 539 - February 24, 2014
- Imprint: Academic Press
- Editor: Jon Lorsch
- Language: English
- Hardback ISBN:9 7 8 - 0 - 1 2 - 4 2 0 1 2 0 - 0
- eBook ISBN:9 7 8 - 0 - 1 2 - 4 2 0 1 7 9 - 8
Laboratory Methods in Enzymology: Protein Part B brings together a number of core protocols concentrating on protein, carefully written and edited by expert… Read more
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Request a sales quoteLaboratory Methods in Enzymology: Protein Part B brings together a number of core protocols concentrating on protein, carefully written and edited by experts.
- Indispensable tool for the researcher
- Carefully written and edited by experts to contain step-by-step protocols
- In this volume we have brought together a number of core protocols concentrating on protein
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
Contributors
Miscellaneous
Preface
Section I: Protein Protocols/Protein In Vitro Translation
Chapter One: In Vitro Synthesis of Proteins in Bacterial Extracts
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 Grow and harvest E. coli for the S30 extract
6 Step 2 Preparation of the S30 extract
7 Step 3 Optimization of the coupled transcription and translation reaction
Chapter Two: Preparation of a Saccharomyces cerevisiae Cell-Free Extract for In Vitro Translation
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 Preparation of Yeast Cell-Free Extract
6 Step 2 Cell-Free Translation
Section II: Protein Protocols/Protein In Vivo Binding Assays
Chapter Three: Yeast Two-Hybrid Screen
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 Small-Scale Transformation of Yeast with pDBLeu-X
6 Step 2 Two-Hybrid Screen
7 Step 3 Confirmation of Positive Interactors
8 Step 4 Plasmid Rescue from Yeast
9 Step 5 Electroporation of E. coli with Yeast DNA and Identification of Positive Interactors
10 Step 6 Back-Transformation of Yeast and Further Confirmation of Interactions
Chapter Four: UV Cross-Linking of Interacting RNA and Protein in Cultured Cells
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 UV Cross-Link RNA–Protein Complexes
6 Step 2 SDS Lysis of Cells
7 Step 3 Immunoprecipitation
8 Step 4 Proteinase K Treatment of RNA Samples
9 Step 5 RNA Analysis
10 Step 6 Protein Analysis
Chapter Five: Analysis of RNA–Protein Interactions by Cell Mixing
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 Cell Mixing
6 Step 2 Cell Lysis
7 Step 3 Immunoprecipitation
8 Step 4 Proteinase K Treatment of RNA Samples
9 Step 5 Northern and Western Blot Analysis
Chapter Six: General Protein–Protein Cross-Linking
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 Calculate the Amount of BS3 to Use
6 Step 2 Protein Cross-Linking
Chapter Seven: Chromatin Immunoprecipitation and Multiplex Sequencing (ChIP-Seq) to Identify Global Transcription Factor Binding Sites in the Nematode Caenorhabditis Elegans
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 Preparation of Extract from Formaldehyde-Fixed Caenorhabditis elegans Embryos and Larvae
6 Step 2 Washes and Collection of the Immunocomplexes and ChIP DNA Purification
7 Step 3 Library Preparation for Multiplex Sequencing Using the Illumina Genome Analyzer
Acknowledgments
Chapter Eight: PAR-CLIP (Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation): a Step-By-Step Protocol to the Transcriptome-Wide Identification of Binding Sites of RNA-Binding Proteins
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 UV Crosslinking of 4-Thiouridine-Labeled Cells (Day 1)
6 Step 2 Preparation of Cell Lysate for Immunoprecipitation (Day 2)
7 Step 3 Preparation of the Magnetic Beads (Day 2)
8 Step 4 Immunoprecipitation and Second RNase T1 Treatment (Day 2)
9 Step 5 Dephosphorylation and Radiolabeling RNA Segments Crosslinked to Immunoprecipitated Proteins (Day 2)
10 Step 6 SDS-PAGE and Electroelution of Cross-Linked RNA-Protein Complexes from Gel Slices (Days 2 and 3)
11 Step 7 Proteinase K Digestion (Day 3)
12 Step 8 3′-Adapter Ligation for cDNA Library Preparation (Day 3 overnight, day 4, beginning of day 5)
13 Step 9 5′-Adapter Ligation for cDNA Library Preparation (Day 5, beginning of day 6)
14 Step 10 cDNA Library Preparation/Reverse Transcription (Day 6)
15 Step 11 PCR Amplification of cDNA Library & Sample Preparation for Sequencing (Day 6)
16 Step 12 Determination of Incorporation Levels of 4SU into Total RNA
Chapter Nine: Determining the RNA Specificity and Targets of RNA-Binding Proteins using a Three-Hybrid System
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 Pilot Transformation to Determine Expected Transformation Efficiency
6 Step 2 Determine 3-AT Concentration to be Used in Selection
7 Step 3 Introduce the Hybrid RNA Library
8 Step 4 Assay β-Galactosidase Activity
9 Step 5 Cure the RNA Plasmid and Test Positives for Protein Dependence
10 Step 6 Isolate Plasmids for Autoactivation Test and Sequencing
11 Step 7 Determine Binding Specificity Using Mutant and Control Proteins
12 Step 8 Functional Tests or Additional Screens
Chapter Ten: Dissecting a Known RNA–Protein Interaction using a Yeast Three-Hybrid System
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1A Assaying Interactions: Qualitative Filter Assay for β-Galactosidase Activity
6 Step 1B Assaying Interactions: Quantitative Solution Assay for β-Galactosidase Activity
7 Step 1C Assaying Interactions: 3-Aminotriazole (3-AT) Resistance Assay
Chapter Eleven: Identifying Proteins that Bind a Known RNA Sequence Using the Yeast Three-Hybrid System
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1 Pilot Transformation to Determine Expected Transformation Efficiency
6 Step 2 Determine 3-AT Concentration to be Used in Selection
7 Step 3 Introduce the cDNA Library
8 Step 4 Eliminate RNA-Independent False Positives by Colony Color
9 Step 5 Assay β-Galactosidase Activity
10 Step 6 Cure the RNA Plasmid and Test Positives for RNA Dependence
11 Step 7 Isolate Plasmids for Autoactivation Test and Sequencing
12 Step 8 Determine Binding Specificity Using Mutant and Control RNAs
13 Step 9 Functional Tests or Additional Screens
Author Index
Subject Index
- Edition: 1
- Volume: 539
- Published: February 24, 2014
- No. of pages (Hardback): 240
- No. of pages (eBook): 240
- Imprint: Academic Press
- Language: English
- Hardback ISBN: 9780124201200
- eBook ISBN: 9780124201798
JL
Jon Lorsch
Affiliations and expertise
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD, USARead Laboratory Methods in Enzymology: Protein Part B on ScienceDirect